Journal: Biology Direct

Article Title: ELK4 induced upregulation of HOMER3 promotes the proliferation and metastasis in glioma via Wnt/β-catenin/EMT signaling pathway

doi: 10.1186/s13062-025-00643-w

Figure Lengend Snippet: Identification of transcription factors regulating HOMER3 expression in glioma. ( A ) Prediction of transcription factor binding sites in the HOMER3 promoter using JASPAR, highlighting ELK4, SP1, and CREB1 as potential regulators. ( B ) Association of expression levels of SP1, CREB1, and ELK4 with the overall survival of glioma patients from TCGA datasets. ( C ) Expression patterns of SP1 and ELK4 in LGG and GBM tissues compared to normal tissues. ( D ) ELK4 binding site predictions on the HOMER3 promoter illustrated. ( E ) Chromatin immunoprecipitation (ChIP) analysis confirming ELK4 binding to the HOMER3 promoter. ( F ) Luciferase reporter assays demonstrating increased luciferase activity in the presence of wild-type ELK4 binding sites in U251 and U87 glioma cells. ( G ) Elevated expression levels of ELK4 in glioma cell lines compared to normal cells as assessed by RT-PCR and western blot analysis. ( H ) Knockdown of ELK4 via shRNAs, which was confirmed by RT-PCR and western bot. ( I and J ) The effect of ELK4 knockdown on glioma cell proliferation assessed by CCK-8 and clonogenic assays. ( K and L ) In vivo analysis of ELK4 knockdown effects on tumor growth. Each group contained n = 6 mice, and the experiment was repeated three times independently. ( M ) Western blot for the expression of CDK4 and CDK6 after ELK4 knockdown. ( N ) The effects of ELK4 knockdown on the migration and invasion of U251 and U87 cells. ( O ) The effects of ELK4 knockdown on the expression of EMT-related proteins by western blot. All experiments were performed in three independent biological replicates ( n = 3) to ensure the reproducibility and reliability of the results

Article Snippet: The following step was to extract the DNA fragments linked to ELK4 from the lysate using an anti-ELK4 antibody(Cat no: 14666-1-AP, Proteintech, Wuhan, China).

Techniques: Expressing, Binding Assay, Chromatin Immunoprecipitation, Luciferase, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Knockdown, CCK-8 Assay, In Vivo, Migration

Journal: Brain research bulletin

Article Title: ELK4 ameliorates cognitive impairment and neuroinflammation induced by obstructive sleep apnea.

doi: 10.1016/j.brainresbull.2024.111054

Figure Lengend Snippet: Fig. 1. ELK4 expression is significantly reduced in IH-induced microglia and brain tissues of mice with OSA. (A) Transcriptome differences in peripheral blood mononuclear cells from 8 normal subjects and 16 patients with OSA syndrome. (B) Intersection of the human transcription factors list in the HumanTFDB database against DEGs screened in the GSE61463 dataset. (C) ELK4 mRNA expression in microglia induced with IH or not detected by RT-qPCR (t = 10.59, df = 4, p = 0.0004). (D) ELK4 protein expression in microglia induced with IH or not detected by western blot analysis (t = 31.80, df = 4, p < 0.0001). (E) ELK4 mRNA expression in the brain tissues of mice induced with OSA or not detected by RT-qPCR (t = 7.970, df = 8, p < 0.0001). (F) ELK4 protein expression in the brain tissues of mice induced with OSA or not detected by western blot analysis (t = 10.38, df = 8, p < 0.0001). For C-F, unpaired t-test. Data are expressed as the means ± SEM; n = 5; ***p < 0.001.

Article Snippet: Membranes were blocked in Trisbuffered saline and 5 % skimmed milk for 1 h at room temperature (RM) and incubated overnight at 4◦C using primary antibodies against ELK4 (1/800; 14666–1-AP; ProteinTech Group, Chicago, IL, USA), FNDC5 (1/3000; ab174833; Abcam), and GAPDH (1/5000; ab181602; Abcam).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Brain research bulletin

Article Title: ELK4 ameliorates cognitive impairment and neuroinflammation induced by obstructive sleep apnea.

doi: 10.1016/j.brainresbull.2024.111054

Figure Lengend Snippet: Fig. 2. Overexpression of ELK4 inhibits pro-inflammatory activation of microglia to ameliorate OSA-associated brain injury. (A) The mean time of escape latency during 5 days of training (F (3, 80) = 619.6, p < 0.0001), the mean percentage of time in the target quadrant (F (3, 16) = 528.4, p < 0.0001), and mean number of mouse platform crossings on day 6 (F (3, 16) = 50.13, p < 0.0001) in the MWM test. (B) The levels of IL-1β (F (3, 16) = 15.67, p < 0.0001), IL-6 (F (3, 16) = 18.99, p < 0.0001), TNF-α (F (3, 16) = 16.49, p < 0.0001), IL-10 (F (3, 16) = 15.76, p < 0.0001), TGF-β1 (F (3, 16) = 15.32, p < 0.0001), and BDNF (F (3, 16) = 18.88, p < 0.0001) in the hippocampal tissues of mice were examined using ELISA. (C) The levels of oxidative stress markers MDA (F (3, 16) = 16.74, p < 0.0001), SOD (F (3, 16) = 22.29, p < 0.0001), and GSH (F (3, 16) = 39.73, p < 0.0001) in the hippocampal tissues of mice were examined. (D) The neuronal pathomorphology in the brain tissues of mice was observed using HE staining. (E) Apoptosis in neurons in the brain tissues of mice was observed using TUNEL (F (3, 16) = 58.04, p < 0.0001). (F) Microglial activation in the brain tissues of mice was observed using immunofluorescence staining of Iba1 (F (3, 16) = 47.94, p < 0.0001). For B-F, one-way ANOVA; for A-1, two-way ANOVA. Data are expressed as the means ± SEM; n = 5; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Membranes were blocked in Trisbuffered saline and 5 % skimmed milk for 1 h at room temperature (RM) and incubated overnight at 4◦C using primary antibodies against ELK4 (1/800; 14666–1-AP; ProteinTech Group, Chicago, IL, USA), FNDC5 (1/3000; ab174833; Abcam), and GAPDH (1/5000; ab181602; Abcam).

Techniques: Over Expression, Activation Assay, Enzyme-linked Immunosorbent Assay, Staining, TUNEL Assay, Immunofluorescence

Journal: Brain research bulletin

Article Title: ELK4 ameliorates cognitive impairment and neuroinflammation induced by obstructive sleep apnea.

doi: 10.1016/j.brainresbull.2024.111054

Figure Lengend Snippet: Fig. 3. Overexpression of ELK4 inhibits the level of inflammation and oxidative stress in BV2 cells. The BV2 cells were infected with oe-NC or oe-ELK4 and subjected to IH modeling. (A) Microglia M1/M2 phenotype was determined using flow cytometry (F (3, 8) = 80.62, p < 0.0001). (B) The levels of IL-1β (F (3, 8) = 14.37, p = 0.0014), IL-6 (F (3, 8) = 12.64, p = 0.0021), TNF-α (F (3, 8) = 16.15, p = 0.0009), IL-10 (F (3, 8) = 26.20, p = 0.0002), TGF-β1 (F (3, 8) = 23.02, P = 0.0003), and BDNF (F (3, 8) = 16.15, p = 0.0009) in BV2 cells were examined using ELISA. (C) The levels of oxidative stress markers MDA (F (3, 8) = 48.88, p < 0.0001), SOD (F (3, 8) = 45.00, p < 0.0001), and GSH (F (3, 8) = 38.03, p < 0.0001) in BV2 cells were examined. For A-C, one-way ANOVA. Data are representative of at least 3 independent experiments and presented as mean ± SEM in all assays; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Membranes were blocked in Trisbuffered saline and 5 % skimmed milk for 1 h at room temperature (RM) and incubated overnight at 4◦C using primary antibodies against ELK4 (1/800; 14666–1-AP; ProteinTech Group, Chicago, IL, USA), FNDC5 (1/3000; ab174833; Abcam), and GAPDH (1/5000; ab181602; Abcam).

Techniques: Over Expression, Infection, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Brain research bulletin

Article Title: ELK4 ameliorates cognitive impairment and neuroinflammation induced by obstructive sleep apnea.

doi: 10.1016/j.brainresbull.2024.111054

Figure Lengend Snippet: Fig. 4. ELK4 transcriptionally promotes FNDC5 expression by binding to the promoter of FNDC5. The correlation between ELK4 and ALDH1A3 (A), FNDC5 (B), MEPCE (C), NAA50 (D), and RSRC2 (E) in the brain tissues in the GEPIA database. (F) The binding sites between ELK4 and the FNDC5 promoter were predicted in the JASPAR website. (G) FNDC5 mRNA and protein expression in BV2 cells with different treatments were examined using RT-qPCR (F (3, 8) = 55.66, p < 0.0001) and western blot analysis (F (3, 8) = 129.1, p < 0.0001). (H) The enrichment of FNDC5 promoter fragments pulled by anti-ELK4 was examined using ChIP (t = 13.53, df = 4, p = 0.0002). (I) Luciferase activity in BV2 cells with different treatments was examined using a dual-luciferase activity assay (F (3, 8) = 72.87, p < 0.0001). (J) Irisin content in BV2 cells with different treatments was examined using ELISA (F (3, 8) = 19.79, p = 0.0005). For G and I-J, one-way ANOVA; for H, unpaired t-test. Data are representative of at least 3 independent experiments and presented as mean ± SEM in all assays; **p < 0.01, ***p < 0.001.

Article Snippet: Membranes were blocked in Trisbuffered saline and 5 % skimmed milk for 1 h at room temperature (RM) and incubated overnight at 4◦C using primary antibodies against ELK4 (1/800; 14666–1-AP; ProteinTech Group, Chicago, IL, USA), FNDC5 (1/3000; ab174833; Abcam), and GAPDH (1/5000; ab181602; Abcam).

Techniques: Expressing, Binding Assay, Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Brain research bulletin

Article Title: ELK4 ameliorates cognitive impairment and neuroinflammation induced by obstructive sleep apnea.

doi: 10.1016/j.brainresbull.2024.111054

Figure Lengend Snippet: Fig. 5. Knockdown of FNDC5 reverses the protective effect of ELK4 on OSAS-related neuroinflammation and cognitive dysfunction. (A) The mean time of escape latency during 5 days of training (F (1, 40) = 469.4, p < 0.0001), mean percentage of time in the target quadrant (t = 21.49, df = 8, p < 0.0001), and mean number of mouse platform crossings on day 6 (t = 4.802, df = 8, p = 0.0014) in the MWM test. (B) The levels of IL-1β (t = 3.102, df = 8, p = 0.0146), IL-6 (t = 3.713, df = 8, p = 0.0059), TNF-α (t = 3.540, df = 8, p = 0.0076), IL-10 (t = 3.452, df = 8, p = 0.0087), TGF-β1 (t = 3.142, df = 8, p = 0.0138), and BDNF (t = 3.309, df = 8, p = 0.0107) in the hippocampal tissues of mice were examined using ELISA. (C) The levels of oxidative stress markers MDA (t = 3.184, df = 8, p = 0.0129), SOD (t = 4.381, df = 8, p = 0.0023), and GSH (t = 4.809, df = 8, p = 0.0013) in the hippocampal tissues of mice were examined. (D) The neuronal pathomorphology in the brain tissues of mice was observed using HE staining. (E) Apoptosis in neurons in the brain tissues of mice was observed using TUNEL (t = 4.674, df = 8, p = 0.0016). (F) Microglial activation in the brain tissues of mice was observed using immunofluorescence staining of Iba1 (t = 7.648, df = 8, p < 0.0001). (G) Irisin contents in the hippocampal tissues of mice were examined using ELISA (F (5, 24) = 27.65, p < 0.0001). For A-C and E-F, unpaired t-test; for G, one-way ANOVA; for A-1, two- way ANOVA. Data are expressed as the means ± SEM; n = 5; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Membranes were blocked in Trisbuffered saline and 5 % skimmed milk for 1 h at room temperature (RM) and incubated overnight at 4◦C using primary antibodies against ELK4 (1/800; 14666–1-AP; ProteinTech Group, Chicago, IL, USA), FNDC5 (1/3000; ab174833; Abcam), and GAPDH (1/5000; ab181602; Abcam).

Techniques: Knockdown, Enzyme-linked Immunosorbent Assay, Staining, TUNEL Assay, Activation Assay, Immunofluorescence

Journal: Brain research bulletin

Article Title: ELK4 ameliorates cognitive impairment and neuroinflammation induced by obstructive sleep apnea.

doi: 10.1016/j.brainresbull.2024.111054

Figure Lengend Snippet: Fig. 6. Schematic illustrating the mechanism. Reduced ELK4 expression in OSA reduces FNDC5 transcriptional expression, leading to pro-inflammatory activation of microglia and cognitive dysfunction. OSA progression was alleviated by activating ELK4-mediated FNDC5 transcriptional expression.

Article Snippet: Membranes were blocked in Trisbuffered saline and 5 % skimmed milk for 1 h at room temperature (RM) and incubated overnight at 4◦C using primary antibodies against ELK4 (1/800; 14666–1-AP; ProteinTech Group, Chicago, IL, USA), FNDC5 (1/3000; ab174833; Abcam), and GAPDH (1/5000; ab181602; Abcam).

Techniques: Expressing, Activation Assay